The NKG2D stimulatory receptor, expressed by NK cells, CD8+, and other T cells, recognizes self ligands that are poorly expressed by normal cells and upregulated by various tumor cells. Expression of NKG2D ligands by tumor cells sensitizes the cells to lysis by NK cells and in some cases T cells. Our central hypothesis is that NKG2D serves (in part) as a host tumor surveillance apparatus that enables NK cells and T cells to eliminate very early stage tumor cells that upregulate NKG2D ligands as a result of oncogenic stress To address key aspects of this hypothesis, we have generated Nkg2d-/- mice. Studies in the previous funding period demonstrated that Nkg2d-/- mice are impaired in immune surveillance of a highly aggressive, early- arising form of prostate adenocarcinoma in the TRAMP oncogene-transgenic mice, and in surveillance of fibrosarcomas induced by the carcinogene methylcolanthrene. We propose here to investigate several key mechanistic issues of NKG2Ds role in cancer immune-surveillance. Specific Aim 1 will address whether NK cells and/or T cells mediate NKG2D-mediated surveillance in cancer models in vivo. We will use genetic studies combining NKG2D knockout mice with mice deficient for NK cells and/or T cells to determine whether NKG2D surveillance is primarily mediated by NK cells, T cells, or both. Specific Aim 2 will address the role of NKG2D specifically on CD8 T cells in specific antitumor responses in vivo. Because of controversy surrounding the role of NKG2D in enhancing CD8 T cell responses to tumors, we will use defined TCR transgenic T cells from NKG2D knockout or wildtype mice in an adoptive transfer/tumor challenge model, in order to address whether NKG2D expressed by CD8 T cells enhances initial proliferative and functional responses, formation of memory cells, maintenance of functionality in the memory stage and capacity to reject tumors. Specific Aim 3 will determine whether NKG2D-dependent surveillance of TRAMP tumors occurs at the stage of tumor initiation. Our central hypothesis is that NKG2D surveillance is a consequence of NKG2D ligand upregulation resulting from pathways that serve as the earliest barriers to tumorigenesis. It is critical to determine whether surveillance occurs at such an early stage, or later. To address the proposal that NKG2D-dependent surveillance acts at the earliest stages of tumor initiation, or later, we will investigate tumor formation in timed cohorts of wildtype or NKG2D knockout mice, using NKG2D ligand expression patterns, histopathology analysis, and analysis of immune infiltrates as methods to pinpoint the stage at which surveillance occurs and its relation to ligand expression and immune cell infiltration. This comprehensive series of experiments will definitively test the role of NKG2D in NK cells and T cells, their interactions, the influence of NKG2D on CD8 T cell responses, and whether NKG2D acts on early or precancerous cells as opposed to more advanced tumors. There is no doubt that the conclusions of these studies will provide fundamental understanding of NKG2Ds role. This research addresses how the immune response attacks cancer cells. We are testing the hypothesis that a specific receptor protein called NKG2D on the surface of immune cells enables these cells to attack and kill cancer cells. The preliminary results show that animals that lack the protein due to a mutation have a higher incidence of cancer. Yet some cancer cells escape recognition by this recognition system. The results of our results will help to guide the design of therapeutic agents that help our immune systems attack cancer.